CN102174383A - DNA (deoxyribonucleic acid) biosensor chip based on nanometer magnetic bead technique and experimental method thereof - Google Patents
DNA (deoxyribonucleic acid) biosensor chip based on nanometer magnetic bead technique and experimental method thereof Download PDFInfo
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- CN102174383A CN102174383A CN2011100378774A CN201110037877A CN102174383A CN 102174383 A CN102174383 A CN 102174383A CN 2011100378774 A CN2011100378774 A CN 2011100378774A CN 201110037877 A CN201110037877 A CN 201110037877A CN 102174383 A CN102174383 A CN 102174383A
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Abstract
On a conventional T-shaped glass chip, a permanent magnet is utilized to fix and decorate a nanometer magnetic bead of a DNA probe in a separation channel of a chip; the rapid, sensitive, high-specific, high-stability, low-cost separation detection on the DNA is realized by utilizing electric drive sampling and combining a laser induced fluorescence detector or an electrochemical detector, thus solving the defects that the time of separating and detecting the DNA by utilizing a microfluidic chip is long, the separation effect is poor, the sensitivity is low, the specific is poor and the stability is poor and the like; the interface manufacturing is avoided by an electric sampling manner; the influence of dead volume is removed; and the sample use amount is reduced in a narrower and uniform micro pipeline, and meanwhile the lower detection limit is ensured.
Description
Technical field
The present invention relates to biologic sensor chip of a kind of chip separation detection DNA and preparation method thereof, particularly a kind of micro-fluidic DNA biologic sensor chip based on the nanometer magnetic bead technology and preparation method thereof.
Background technology
At present, the common method based on chip separation detection DNA has chip gel electrophoresis and chip Non-gel Sieving Electrophoresis.There are loaded down with trivial details consuming time, the shortcomings such as column life short, easy obstruction of gel column preparation process in the former; The latter's non-glue medium system selects still do not have clear and definite rule, and the existence of non-glue medium may influence follow-up detection.In addition, the both exist separating pipe long, can't separate the DNA of identical base number and can't distinguish shortcoming such as single base mismatch.
In recent years, the chip that has the nanotubes structure was used as separation and the research of DNA, but its cleanliness factor to pipeline manufacture craft and environment is had higher requirement.Simultaneously, along with the development of DNA biosensor, the development of DNA biologic sensor chip also receives more and more scholars' concern.At present, existing investigator attempts integrated microelectrode system on chip, utilizes that enrichment with magnetic bead target dna and signal probe are laggard goes into microchannel, detects with electrochemical signals.But, because the microelectrode making is loaded down with trivial details relatively, the processing requirement height, the cost height can't be popularized.In addition, someone attempts by the fixing magnetic nanoparticle in the microchannel of permanent magnet, transport DNA with pressure pump, realize separation, enrichment and the detection of DNA, but its some chip pipeline configuration is complicated, and some is the microchannel broad of magnetic bead fixedly, influences detectability, and the interface between chip and the pressure pump is made trouble, and the pressure-controlling micrometeor is accurate inadequately.
Summary of the invention
The object of the present invention is to provide a kind of DNA biologic sensor chip based on the nanometer magnetic bead technology, use detection that this chip carries out DNA with separate have fast, sensitivity, high specific, high stability and characteristics cheaply.
Technical program of the present invention lies in: a kind of DNA biologic sensor chip based on the nanometer magnetic bead technology, comprise cover plate glass and substrate glass, it is characterized in that: be etched with T type microchannel on the described substrate glass, cover plate sample pool, waste liquid pool and the Buffer Pool of being respectively equipped with on glass at three end points places of corresponding T type microchannel, described sample pool is provided with a pipeline, the pipeline the other end is connected with peristaltic pump, and the microchannel between described sample pool and the waste liquid pool is provided with button magnet.
Another object of the present invention is to provide a kind of experimental technique of the DNA biologic sensor chip based on the nanometer magnetic bead technology, this method is convenient to operation, and DNA isolation is quick, sensitive.
Another technical scheme of the present invention is: a kind of experimental technique of the DNA biologic sensor chip based on the nanometer magnetic bead technology is characterized in that: carry out according to the following steps:
1) plastic conduit that is provided with on the sample pool is realized being tightly connected with glue and sample pool, the other end of plastic conduit connects goes up peristaltic pump, twitch by peristaltic pump produces negative pressure, the magnetic nanoparticle that is connected with dna probe in the waste liquid pool is introduced T type pipeline, A position on the T type pipeline is provided with button magnet, and the magnetic nanoparticle that is connected with dna probe is fixed on the A position;
2) between sample pool and waste liquid pool, add voltage, utilize electric drive mode to bring target DNA into T type pipeline, realize sample introduction;
3) between Buffer Pool and waste liquid pool, add voltage, drive the ducted target DNA band of T type to waste liquid pool by electricity, when flowing through the A position, fixed dna probe generation hybridization on target DNA and the magnetic nanoparticle, target DNA is attracted on the magnetic nanoparticle;
4) remove button magnet, magnetic nanoparticle moves to the waste liquid pool direction under electrophoretic effect, by laser induced fluorescence detector or electrochemical detector the DNA on the magnetic nanoparticle is detected again.
The invention has the advantages that: routine " on the T type glass-chip; utilize permanent magnet in the split tunnel of chip, fixedly to modify the nanometer magnetic bead of dna probe; utilize electricity to drive sample introduction; in conjunction with laser induced fluorescence detector or electrochemical detector realize to DNA fast; sensitivity; high specific, high stability and separation detection cheaply, it is long to have solved the micro-fluidic chip separation detection DNA time, inferior separating effect, sensitivity is low, shortcoming such as poor specificity and poor stability, the mode of electrokinetic injection has been avoided the making of interface, eliminated the influence of dead volume, narrower and uniform microchannel guarantees lower detectability in the consumption that reduces sample.
Description of drawings
Fig. 1 is a structural representation of the present invention.
Embodiment
A kind of DNA biologic sensor chip based on the nanometer magnetic bead technology, comprise cover plate glass and substrate glass, it is characterized in that: be etched with T type microchannel 8 on the described substrate glass, cover plate sample pool 1, waste liquid pool 2 and the Buffer Pool 3 of being respectively equipped with on glass at three end points places of corresponding T type microchannel, described sample pool is provided with a pipeline 4, the pipeline the other end is connected with peristaltic pump 5, and the microchannel between described sample pool and the waste liquid pool is provided with button magnet 9.
Magnetic nanoparticle 6 is housed in the described waste liquid pool, is connected with dna probe 7 on the described magnetic nanoparticle.
A kind of experimental technique of the DNA biologic sensor chip based on the nanometer magnetic bead technology is characterized in that: carry out according to the following steps:
1) plastic conduit that is provided with on the sample pool is realized being tightly connected with glue and sample pool, the other end of plastic conduit connects goes up peristaltic pump, twitch by peristaltic pump produces negative pressure, the magnetic nanoparticle that is connected with dna probe in the waste liquid pool is introduced T type pipeline, A position on the T type pipeline is provided with button magnet, and the magnetic nanoparticle that is connected with dna probe is fixed on the A position;
2) between sample pool and waste liquid pool, add voltage, utilize electric drive mode to bring target DNA into T type pipeline, realize sample introduction;
3) between Buffer Pool and waste liquid pool, add voltage, drive the ducted target DNA band of T type to waste liquid pool by electricity, when flowing through the A position, fixed dna probe generation hybridization on target DNA and the magnetic nanoparticle, target DNA is attracted on the magnetic nanoparticle;
4) remove button magnet, magnetic nanoparticle moves to the waste liquid pool direction under electrophoretic effect, by laser induced fluorescence detector or electrochemical detector the DNA on the magnetic nanoparticle is detected again.
Described substrate and cover plate all adopt TIO glass.
The width of described T type microchannel is 40 ~ 60 microns, and the degree of depth is 10 ~ 15 microns
The preparation method of the DNA biologic sensor chip based on the nanometer magnetic bead technology of the present invention is as follows: 1) etching T type microchannel on substrate glass; 2) three end points places of corresponding substrate T type microchannel use glass drill respectively to get out a hole as sample pool, waste liquid pool and Buffer Pool on cover plate; 3) by high temperature substrate and cover plate are bonded to required chip.
Described substrate and cover plate all adopt TIO glass, are convenient to draw materials, and the cost of material is low.
Lithographic method described in the step 1) adopts photoetching technique and wet etching, and aforesaid method is the common method of glass etching, need not special process, and is easy and simple to handle.
The microchannel width that forms in the step 1) is 40 ~ 60 microns, and the degree of depth is 10 ~ 15 microns.
Step 2) hole that forms on the cover plate in is the hole of diameter 3mm.
The above only is preferred embodiment of the present invention, and all equalizations of being done according to the present patent application claim change and modify, and all should belong to covering scope of the present invention.
Claims (5)
1. DNA biologic sensor chip based on the nanometer magnetic bead technology, comprise cover plate glass and substrate glass, it is characterized in that: be etched with T type microchannel on the described substrate glass, cover plate sample pool, waste liquid pool and the Buffer Pool of being respectively equipped with on glass at three end points places of corresponding T type microchannel, described sample pool is provided with a pipeline, the pipeline the other end is connected with peristaltic pump, and the microchannel between described sample pool and the waste liquid pool is provided with button magnet.
2. the DNA biologic sensor chip based on the nanometer magnetic bead technology according to claim 1 is characterized in that: in the described waste liquid pool magnetic nanoparticle is housed, is connected with dna probe on the described magnetic nanoparticle.
3. experimental technique based on the DNA biologic sensor chip of nanometer magnetic bead technology is characterized in that: carry out according to the following steps:
1) plastic conduit that is provided with on the sample pool is realized being tightly connected with glue and sample pool, the other end of plastic conduit connects goes up peristaltic pump, twitch by peristaltic pump produces negative pressure, the magnetic nanoparticle that is connected with dna probe in the waste liquid pool is introduced T type pipeline, A position on the T type pipeline is provided with button magnet, and the magnetic nanoparticle that is connected with dna probe is fixed on the A position;
2) between sample pool and waste liquid pool, add voltage, utilize electric drive mode to bring target DNA into T type pipeline, realize sample introduction;
3) between Buffer Pool and waste liquid pool, add voltage, drive the ducted target DNA band of T type to waste liquid pool by electricity, when flowing through the A position, fixed dna probe generation hybridization on target DNA and the magnetic nanoparticle, target DNA is attracted on the magnetic nanoparticle;
4) remove button magnet, magnetic nanoparticle moves to the waste liquid pool direction under electrophoretic effect, by laser induced fluorescence detector or electrochemical detector the DNA on the magnetic nanoparticle is detected again.
4. the DNA biologic sensor chip based on the nanometer magnetic bead technology according to claim 1, it is characterized in that: described substrate and cover plate all adopt TIO glass.
5. the DNA biologic sensor chip based on the nanometer magnetic bead technology according to claim 1 is characterized in that: the width of described T type microchannel is 40 ~ 60 microns, and the degree of depth is 10 ~ 15 microns.
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| CN2011100378774A CN102174383A (en) | 2011-02-15 | 2011-02-15 | DNA (deoxyribonucleic acid) biosensor chip based on nanometer magnetic bead technique and experimental method thereof |
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Cited By (11)
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| CN102854304A (en) * | 2012-07-06 | 2013-01-02 | 武汉大学 | Pathogen detection method based on micro-fluidic chip |
| CN103616523A (en) * | 2012-12-31 | 2014-03-05 | 烟台卓越生物技术有限责任公司 | Improved type detection card of portable type biochemical detector |
| CN103901189A (en) * | 2014-04-16 | 2014-07-02 | 北京化工大学 | Automatic immunodetection micro-fluidic chip fluorescent quantification detection device |
| CN104316680A (en) * | 2014-11-04 | 2015-01-28 | 张晓杰 | Detection device of helicobacter pylori and application method thereof |
| CN104540589A (en) * | 2012-06-15 | 2015-04-22 | 通用电气公司 | Microfluidic system, cartridge and method for preparing sample |
| CN104614521A (en) * | 2015-02-11 | 2015-05-13 | 清华大学 | Immune agglomeration detection method, chip and system based on micro-fluidic chip |
| CN104697841A (en) * | 2015-03-30 | 2015-06-10 | 北京热景生物技术有限公司 | Magnetic particle separating and transferring device and method and application thereof |
| CN105074465A (en) * | 2013-02-22 | 2015-11-18 | 株式会社日立高新技术 | Biological analyzing device and biomolecule analyzing equipment |
| CN105891493A (en) * | 2016-06-01 | 2016-08-24 | 章健 | Fluorescence detection device and detection method thereof |
| CN108579823A (en) * | 2018-05-18 | 2018-09-28 | 江苏赛威分离科技有限公司 | A kind of technique run close to resin bed using distributor |
| CN109270259A (en) * | 2018-09-12 | 2019-01-25 | 南方医科大学 | A kind of endotoxic method of detection |
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Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104540589A (en) * | 2012-06-15 | 2015-04-22 | 通用电气公司 | Microfluidic system, cartridge and method for preparing sample |
| CN102854304A (en) * | 2012-07-06 | 2013-01-02 | 武汉大学 | Pathogen detection method based on micro-fluidic chip |
| CN102854304B (en) * | 2012-07-06 | 2015-09-16 | 武汉大学 | A kind of pathogen detection method based on micro-fluidic chip |
| CN103616523A (en) * | 2012-12-31 | 2014-03-05 | 烟台卓越生物技术有限责任公司 | Improved type detection card of portable type biochemical detector |
| CN105074465A (en) * | 2013-02-22 | 2015-11-18 | 株式会社日立高新技术 | Biological analyzing device and biomolecule analyzing equipment |
| CN105074465B (en) * | 2013-02-22 | 2017-03-29 | 株式会社日立高新技术 | Bioanalysis device and biomolecule analyzer |
| CN103901189A (en) * | 2014-04-16 | 2014-07-02 | 北京化工大学 | Automatic immunodetection micro-fluidic chip fluorescent quantification detection device |
| CN103901189B (en) * | 2014-04-16 | 2015-12-02 | 北京化工大学 | Immune detection micro-fluidic chip fluorescent quantitation automatic detection device |
| CN104316680A (en) * | 2014-11-04 | 2015-01-28 | 张晓杰 | Detection device of helicobacter pylori and application method thereof |
| CN104316680B (en) * | 2014-11-04 | 2016-08-17 | 张晓杰 | The detection device of a kind of helicobacter pylori and using method thereof |
| CN104614521A (en) * | 2015-02-11 | 2015-05-13 | 清华大学 | Immune agglomeration detection method, chip and system based on micro-fluidic chip |
| CN104697841B (en) * | 2015-03-30 | 2021-01-12 | 北京热景生物技术股份有限公司 | Magnetic particle separation and transfer device, method and application thereof |
| CN104697841A (en) * | 2015-03-30 | 2015-06-10 | 北京热景生物技术有限公司 | Magnetic particle separating and transferring device and method and application thereof |
| CN105891493A (en) * | 2016-06-01 | 2016-08-24 | 章健 | Fluorescence detection device and detection method thereof |
| CN105891493B (en) * | 2016-06-01 | 2017-10-24 | 章健 | A kind of fluorescence detection device and its detection method |
| CN108579823A (en) * | 2018-05-18 | 2018-09-28 | 江苏赛威分离科技有限公司 | A kind of technique run close to resin bed using distributor |
| CN108579823B (en) * | 2018-05-18 | 2021-05-14 | 江苏赛威分离科技有限公司 | Process for operating by adopting distributor close to resin bed layer |
| CN109270259A (en) * | 2018-09-12 | 2019-01-25 | 南方医科大学 | A kind of endotoxic method of detection |
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Application publication date: 20110907 |